Continued efforts have been made to delineate the genetic mechanisms underlying virulence of rotavirus in the newborn gnotobiotic piglet model. A single gene substitution reassortant that had the 1st, 2nd, 5th, 6th, 7th, 8th, or 11th gene from human rotavirus DS-1 strain (which does not induce productive infection or diarrhea in gnotobiotic piglets) and the remaining ten genes from porcine rotavirus SB-1A strain (which consistently induces diarrhea in piglets) induced diarrhea in piglets that was associated with virus shedding. However, piglets challenged with a single gene substitution reassortant that had the VP3 gene, or VP4 gene, or VP7 gene, or NS-28 gene of DS-1 virus and the remaining ten genes from porcine SB-1A virus failed to develop illness or shed virus. Thus, we demonstrated that the 3rd, 4th, 9th (VP7), or 10th (NS28) gene each play an important role in virulence of rotavirus infection in piglets. Piglets challenged with a rotavirus reassortant that had the 3rd, 4th, 9th, and 10th genes of SB-1A virus and the remaining seven genes from DS-1 virus developed diarrhea and shed virus.